Asymmetric Dimethylarginine Protects Neurons from Oxygen Glucose Deprivation Insult by Modulating Connexin36 Expression

Background . Asymmetric dimethylarginine (ADMA) is a nonselective nitric oxide synthase inhibitor. ADMA is thought to inhibit the production of nitric oxide (NO) by neurons after oxygen-glucose deprivation (OGD). The gap junction protein Connexin-36 (cx-36) is involved in the pathophysiology of stroke. We investigated whether ADMA could protect neurons from OGD insults by regulating the expression of cx-36. Methods . Cultured rat cortical neuronal cells were used. Neurons were treated with OGD with or without ADMA pretreatment. The lactate dehydrogenase (LDH) release rate was used to assess neuronal injury. Intracellular NO levels were determined using 4-amino-5-methylamino-2 ′ ,7 ′ -di ﬂ uoro ﬂ uorescein diacetate. Western blotting was performed to detect cx-36 expression. Results . The LDH release rate increased in the supernatant of neurons after the OGD insult, whereas ADMA treatment reduced the LDH release rate. Intracellular NO levels increased following OGD treatment, and this increase was not inhibited by ADMA treatment. Expression of cx-36 was upregulated in neurons under OGD conditions, and treatment with ADMA downregulated the expression of cx-36. Conclusions . ADMA protects neurons from OGD insult, and cx-36 downregulation may be a possible pathway involved in ADMA-mediated neuronal protection.


Introduction
Asymmetric dimethylarginine (ADMA), an endogenous analog of arginine, was found to inhibit nitric oxide (NO) production by competing for the binding site of arginine in nitric oxide synthase (NOS). It nonselectively inhibits NOS, including endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) [1].
ADMA is used as a biomarker to identify endothelial dysfunction owing to its effect on eNOS. Increased ADMA levels have been observed in patients with hypertension, hyperlipidemia, diabetes, atrial fibrillation, old age, and atherosclerotic burden [2][3][4], as well as in patients with acute stroke [5,6] and chronic stroke [7,8]. In addition to inhibition of eNOS, ADMA can also inhibit the activity of iNOS and nNOS. NO produced by iNOS and nNOS may induce oxidative stress in neurons. It is well known that NO production significantly increases after acute ischemic stroke, thereby inducing oxidative stress, which contributes to sec-ondary neuronal injury after stroke [9]. As a nonselective inhibitor of nNOS and iNOS, ADMA could potentially protect neurons during stroke in a very early phase; however, the literature is sparse in this area.
Gap junctions are important signal transduction pathways that allow small molecules (up to 1,000 daltons) and ions to diffuse from one cell to a neighboring cell [10,11]. Gap junctions open when two hemichannels in adjacent plasma membranes dock with each other. Each hemichannel consists of six connexin subunits. One of these subunits, , is expressed mainly in neurons and retinal cells [12][13][14]. In the physiological state, there is little expression of cx-36 in the mature central nervous system. However, in the context of stroke, inflammation, epilepsy, and trauma, cx-36 expression is increased [15][16][17][18][19]. Amounts of opened gap junctions increase rapidly in oxygen-glucosedeprived neurons, causing ionic dysregulation and possibly contributing to neuronal death [20]. Cx-36 has been proven to play a critical role in the pathophysiology of N-methyl-D-aspartate-(NMDA-) mediated neuronal death, with both cx-36 gene knockout and cx-36-specific inhibition showing a protective effect on neurons from NMDA toxicity [21]. However, the role of cx-36 in neuronal death when neurons undergo oxygen-glucose deprivation (OGD) is unknown, not to mention its role in the neuronal protective effect of ADMA. Although ADMA has been shown to modulate the expression of other connexins, such as cx-43, cx-37, and cx-40 [22], the effect of ADMA on cx-36 is still unknown.
Our study aimed to investigate the protective effect of ADMA on neurons in an OGD model and explore the effect of ADMA on cx-36 in neurons after OGD insult.

Primary Cortical Neuronal Cultures. Adult pregnant
Wistar rats were purchased from Shanghai SLAC Laboratory Animal Company, China. Primary cortical neuronal cultures were obtained from fetal Wistar rat at embryonic day 14 (E14). Rats were placed in a 12 h light-dark cycle, under environmentally controlled conditions (ambient temperature, 22 ± 2°C; humidity, 40%) and with free access to food and water. All experimental processes were implemented according to experimental standards of Fudan University, as well as international ethical guidelines for experimentation on animals. Primary cortical neuronal cultures were prepared as previously described, with slight modification [23]. Briefly, the cerebral cortex was removed; after that, meninges were removed from the cortex. The cerebral cortex tissues were dissociated using 0.25% trypsin for 20 min at room temperature (24 ± 2°C). After titration and resuspension in neurobasal A medium supplemented with B27, the cells were plated on poly L-lysine-coated glass coverslips, 96-well plates, or 100 mm dishes. The culture medium was changed by half every 3 days. Cultured neurons were used to investigate in vitro day 7-10 (DIV 7-10).

Oxygen and Glucose
Deprivation/Reoxygenation. Primary neurons (DIV 7-10) were subjected to OGD/reoxygenation (OGD/R) as previously described, with minor modifications [24]. Briefly, the cells were washed three times with Hank's balanced salt solution (HBSS), incubated in deoxygenated glucose-free DMEM in an anaerobic chamber (Forma anaerobic system, Thermo) with an atmosphere (<0.1% O 2 ) containing 85% N 2 , 10% H 2 , and 5% CO 2 at 37°C, and terminated after 45 min by replacing deoxygenated glucose-free DMEM with original medium in normal incubator for the subsequent 0-24 h reoxygenation (R). Control cells were obtained normally.
2.4. Cytotoxicity Assessment. Neuronal cytotoxicity was assessed using a cytotoxicity detection kit ground on the LDH activity released from the cytosol of damaged cells into the supernatant [25]. The experiments were performed according to the manufacturer's protocol. Total LDH activity was measured in the supernatant of cells that had been repeatedly frozen and thawed thrice. The relative LDH release rate is represented as the ratio of extracellular LDH activity to total LDH activity.
2.5. Intracellular NO Assay. Intracellular NO was detected using the fluorescent probe DAF-FM DA [26]. DAF-FM DA penetrates the cell membrane and is converted to DAF-FM by intracellular esterases, which cannot penetrate the cell membrane. The fluorescence emitted by the DAF-FM was very weak; however, the signal became much stronger coexisting together with NO. Neuronal cells were cultured in 96-well plates. The cells on DIV 7-10 after OGD/ R treatment were incubated with DAF-FM DA (5 μM) in an incubator (at 37°C with dark) for 20 min and then rinsed with PBS (pH 7.8) for three times. The DAF-FM fluorescence intensity was detected by a fluorescence microplate reader (SpectraMax M5, Molecular Devices, USA) with an excitation wavelength of 495 nm and an emission wavelength of 515 nm. Intracellular NO production was reflected by the relative ratio to the control cells. The experiments were repeated thrice with eight readings for every group. To differentiate the effect of NOS subtypes, we used nNOS-specific inhibitor 7-NI (with final concentration 50 μM) and iNOS-specific inhibitor 1400w (with final concentration 10 μM).
2.6. Neuronal Cell Pretreatment. ADMA was added to the supernatant of cultured cells in 24 h before OGD/R treatment, and the final concentration was 30 μM. The ADMA concentration remained unchanged throughout the experiment. 7-NI or 1400w was added to the supernatant of cultured cells immediately after OGD treatment.

Western Blot.
Western blotting was used to assay the expression of cx-36. The cells were planted on 100 mm dishes. After diverse treatments, the cells were collected by scraping in ice-cold radioimmunoprecipitation lysis buffer containing 1% Triton, 1% deoxycholate, 0.1% SDS, 1 mmol/l EDTA, 50 mmol/l Tris (pH 7.4), and 150 mmol/l NaCl, supplemented with PMSF, and then centrifuged at 18,000 × g for 20 min. The supernatant was collected. Using the BCA kit, the quantity of the total protein was determined in every supernatant. 10% SDS-PAGE was used to separate the protein electrophoretically. Separated protein was transferred onto PVDF membranes (Millipore). Then, the 2 Oxidative Medicine and Cellular Longevity membranes were immersed in blocking buffer (1× Trisbuffered saline (TBS, pH 7.6) containing 5% BSA) for 2 h at room temperature. The membranes were then incubated overnight with primary cx-36 (diluted 1 : 1000) antibodies at 4°C. After rinsing three times with TBS-Tween (0.1% Tween 20), the membranes were dug into solution of goat anti-mouse secondary HRP-conjugated IgG antibodies (diluted 1 : 1000) for 1 h at room temperature (24 ± 2°C). Equal loading was ensured by detecting the membranes with an anti-mouse β-tubulin (neuronal) antibody (1 : 5,000 dilution). Detection was performed using an enhanced chemiluminescence assay kit (Millipore). The western blots were captured using a Molecular Imager (ChemiDoc™ XRS+, Bio-Rad, USA), and the intensities were quantified using Image Lab software (Bio-Rad, USA).

Statistical
Analysis. Data are presented as mean ± SEM based on three separate experiments. One-way ANOVA analysis was used to analyze the difference between different groups, followed by LSD post hoc tests for multiple comparisons tests or Kruskal-Wallis nonparametric ANOVA with adjustment for multiple comparisons. Paired Student's t-tests were used for comparisons between two groups only. Differences were considered statistically significant at P < 0:05.

Results
3.1. ADMA Protects Neurons from OGD/R Injury. We measured the relative LDH release rate to assess neuronal injury resulting from OGD/R. Compared with those in the control, neurons undergoing OGD injury showed a time-dependent increase in LDH release rate during the time course of reperfusion ( Figure 1). The relative LDH release rate was 46:82 % ± 0:72% of maximal LDH release 24 h after reperfusion. We selected a reperfusion time point of 2 h for further analysis. We found that neurons pretreated with ADMA (0 μM, 3 μM, 10 μM, 30 μM, and 100 μM) showed a decreased LDH release rate after OGD/R. However, we did not observe any concentration-dependent decreases. When pretreated with a high dose of ADMA (300 μM), the LDH release rate remained slightly elevated, although without significance ( Figure 2).

ADMA Did Not
Inhibit the NO Production of Neurons after OGD/R Insult. The NO production in neurons after OGD/R showed a time-dependent profile (Figure 3(a)).
Using the nNOS-specific inhibitor 7-NI and the iNOSspecific inhibitor 1400w, we could easily identify the type of NOS that was the primary source of NO production at different time points after OGD/R. Immediately after OGD/R, NO production markedly increased, which was catalyzed by nNOS (Figure 3(c)). After that, NO production started to decrease until 4 h after OGD/R then started to rise again reaching a peak at 6 h after OGD/R. iNOS was responsible for the second peak in NO production (Figure 3(b)). Surprisingly, as a nonselective NOS inhibitor, ADMA at 30 μM did not suppress NO production in the pretreated neurons. In contrast, the ADMA 30 μM group produced more NO than that by the baseline OGD/R group at 12 h after OGD/R (Figure 3(d)).

ADMA Attenuated the Expression of cx-36 in Neurons
after OGD/R. Since the protective effect of ADMA on neurons after OGD/R was not due to its inhibitory effect on NOS, there must be other mechanisms. Previous studies showed that cx-36 expression increased after ischemic stroke [27] and was accompanied by the transfer of electrolytes from one cell to another, which might lead to the dysfunction and eventual death of cells [20].    Oxidative Medicine and Cellular Longevity the expression of cx-36 increased, which was higher than that of the control and higher than that at 0 h after OGD treatment ( Figure 4). We considered 4 h as the time point for further investigation. Neurons were pretreated with ADMA at 30 μM for 24 h before OGD treatment, as designed previously. The duration of OGD was 45 min. Subsequently, neurons were returned to normal conditions for another 4 h. Our results showed that pretreatment with ADMA at 30 μM attenuated the increased expression of cx-36 4 h after OGD/R ( Figure 5).

Discussion
To the best of our knowledge, this is the first study to focus on the protective effect of ADMA on neurons undergoing an OGD/R insult. Our results showed that ADMA may protect neurons from OGD/R insults. However, intracellular NO level in neurons was not, as we supposed, affected by ADMA following OGD/R insult. Protection might be mediated via the suppression of cx-36 expression.
As mentioned above, ADMA can inhibit nNOS and iNOS, reducing the production of neurotoxic NO, thereby demonstrating its potential neuroprotective ability. Several studies have already proven this hypothesis using different models [28,29]. Cardounel and Zweier showed that ADMA protected neurons from NMDA-induced neurotoxicity [28]. Tang et al. demonstrated that ADMA attenuated ROS accumulation in MPP+-treated PC12 cells and protected neurons from MPP+-triggered cell death [29,30]. However, Wang et al. reported that exogenous ADMA may induce apoptosis in PC12 cells in a concentration-and time-dependent manner [31]. The different effects of ADMA in PC12 cells may be due to differences in the treatment protocols and assessment assays. Zhao et al. found that dimethylarginine dimethylamino hydrolase-1-(DDAH-1-) knockout rats demonstrated aggravated neurological damage after MCAO/R in an in vivo study [32]. This result seems contradictory to that of the present study. As reported previously, DDAH-1 is distributed in multiple cell types, including neurons, astrocyte cells, and vascular structures, while DDAH-2 is distributed exclusively in neurons [33]. As a result, DDAH-1 knockout might affect  Figure 3: Time profile of NO production after OGD/R with different NOS inhibitors. (a) NO production in different time points was higher in neurons after OGD/R than in neurons in normal conditions. (b) NO production in the late stage after OGD/R was inhibited by iNOSspecific inhibitor 1400w, which indicated NO produced in the late stage was mainly produced by iNOS. (c) NO production in the early stage after OGD/R was inhibited by nNOS-specific inhibitor 7-NI, which indicated NO produced in the early stage was mainly produced by nNOS. (d) ADMA 30 μM could not inhibit NO production after OGD/R (n = 3) and * compared with normal condition group, p < 0:05. # Compared with OGD/R group, p < 0:05.

4
Oxidative Medicine and Cellular Longevity various cell types of animals and is not limited to neurons, which could not explain the effect of DDAH-1 knockout in neurons. Our study focused on cortical neurons and provided evidence of the neuroprotective effect of ADMA in cortical neurons undergoing OGD treatment. The different results might lie in different models. It is well known that ADMA is a competitive inhibitor of NOS, where iNOS and nNOS are involved in the physiopathology of stroke. We hypothesized that ADMA protected neurons from OGD/R treatment by attenuating NO levels in neurons. However, we could not prove our hypothesis in this study. We found that the NO levels burst with two peaks in the context of OGD/R (Figure 3(a)). However, exogenous ADMA treatment did not suppress NO (Figure 3(d)). Furthermore, 12 h after OGD/R, NO production increased to a peak level in the OGD/R group with ADMA pretreatment, higher than that in the OGD/R group without ADMA pretreatment. Previous data showed that the nNOS inhibitory effect of ADMA was concentrationdependent. An ADMA concentration of 30 μM might inhibit 23% of NO production by nNOS, while an ADMA concentration of 100 μM could inhibit nNOS activity by 100% [28]. In our study, we used 30 μM as the pretreatment dosage. However, inhibition of nNOS by ADMA was not observed. The possible reasons might be as follows: first, the insult procedures were different in the two studies. In the study by Cardounel and Zweier, neurons were treated with NMDA; as in our study, neurons were treated with OGD/R. The mechanisms underlying the neurotoxic effects of OGD/R are more complex than those of NMDA. Second, the sensitivities of the NO detection assays were different. Third, compared with the specific nNOS inhibitor 7-NI, the nNOS inhibitory effect of ADMA is very weak, and the dosage of ADMA used in our study was not powerful enough to inhibit nNOS. Klein et al. showed that ADMA upregulated iNOS expression in airway epithelial cells in vivo and in vitro [34]; however, the inhibition of iNOS was quite weak as well [35]. ADMA pretreatment might also increase expression of iNOS in neurons. As a result, when the neurons underwent OGD/R, the NO production might appear higher than that without ADMA pretreatment, which might partly explain our data where NO production was higher in group with ADMA pretreatment than those without ADMA treatment at 12 h after OGD/R (Figure 3(d)).
Nevertheless, despite the level of NO in neurons, ADMA still protected neurons from OGD/R ( Figure 2). This is consistent with previous data [28]. Therefore, there may be other mechanisms that enable ADMA-mediated neuronal protection. In the subarachnoid hemorrhage rat model, ADMA was positively correlated with cx-43, indicating that ADMA might regulate the expression of connexins [36].
cx-36 is an electrical synapse that allows the transmission of ions and electricity between neighboring neurons, and cx-36 plaques were found to strengthen gap-junctional conductance rapidly and profoundly [37]. It has been previously shown that cx-36 promotes cortical spreading depolarization and induces ischemic brain damage [38]. Cx-36 may also upregulate the expression of inflammatory cytokines in a depressive-like behavior animal model [39]. As a result, the downregulation or inhibition of cx-36 may have a neuroprotective effect. A transient increase in cx-36 expression was observed in ischemic cortex neurons in vivo and in vitro, and using the cx-36 gene elimination technique and a cx-36 inhibitor, cx-36 was proven to be toxic in stroke [40]. In the case of OGD/R, cx-36 expression in neurons increased. This is consistent with previous in vitro studies that were not limited to cortical neurons [41,42]. According to a study performed by Li     Oxidative Medicine and Cellular Longevity 36 might be the target protein for the protective effect of leonurine in OGD PC12 cells [43]. Moreover, cx-36 inhibition might reduce the level of phosphorylated calmodulindependent protein kinase II (pCaMKII) and the ratio of pCaMKII/CaMKII, thus protecting PC-12 cells from OGD insult [43]. Similar results were observed in the present study. When pretreated with ADMA, the increased expression of cx-36 was attenuated in OGD/R neurons, indicating that ADMA might protect neurons from OGD/R by modulating the cx-36 expression.
The limitation of our study was that we did not use a cx-36 specific inhibitor. It has been shown that cx-36 inhibition is neuroprotective [40,44]. We did not know the mechanism underlying the effect of ADMA on cx-36 expression in this work. According to previous studies, the potential regulator of cx-36 expression or function included CaMKII pathway [45], group II metabotropic glutamate receptors (mGluRs) pathway, and cAMP-dependent protein kinase-dependent signaling pathway [40]. However, we have no idea whether these pathways are also involved in the regulation of ADMA on cx-36, and we also do not know if there is any other pathway. To make the mechanism clear, further investigation is needed.
In our work, we proposed a possibility of ADMA to be a neuroprotective agent in the early stage of stroke. Pretreatment in the study did restrict the clinical application in the current situation. However, situation in human body is quite different from animals, let alone cultured cells. To achieve the goal of translation from bench to bed, lots of work need to be done in the future.
In conclusion, ADMA may protect neurons from OGD/ R insult by attenuating the cx-36 expression. Therefore, ADMA might be a potential agent to protect neurons from death in the early stages of stroke, which requires further experimental and clinical investigation.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.